DNA preservation & DNA extraction protocol for field collection of coral samples suitable for host-, marker gene-, and metagenomics-based sequencing approaches

1 DNA preservation & DNA extraction protocol for field collection of coral samples suitable for host-, marker gene-, and metagenomics-based sequencing approaches Christian R Voolstra*, Luigi Colin, Melanie Dörr, Gabriela Perna, Anna Fiesinger, Anny Cardénas Department of Biology, University of Konstanz, Konstanz, Germany *Corresponding author, [email protected] Version 1.4, release: 11.04.2025 Changes to previous version: - Added extra wash step prior to homogenization of metagenomic samples - Added Symbiodiniaceae pelleting step prior to first cell lysis to minimize eukaryotic DNA carryover - Made the first bead-beating step (to crack open Symbiodiniaceae cells) optional. Summary The ability to collect samples suitable for DNA extraction in remote field settings without the necessity of freezing is a common need for molecular ecologists that has been addressed by the so-called DESS buffer, a solution containing DMSO, EDTA, and saturated NaCl (Dawson, Raskoff, & Jacobs, 1998; Seutin, White, & Boag, 1991). DESS is commonly used as a storage buffer for coral samples (Pinzón et al., 2013; Voolstra et al., 2021; Ziegler et al., 2017) and has also been shown to faithfully preserve DNA for bacterial community analyses (Gray, Pratte, & Kellogg, 2013; Lee, Adams, & Klassen, 2019). What is lesser known is that DESS buffer has been shown to preserve nematode morphology, comparable to formalin-based fixatives (Yoder et al., 2006) and, as such, preserves cell integrity. This makes it suitable for metagenomics applications, which typically rely on differentially lysis of eukaryotic (host) and prokaryotic (microbiome) cells to effectively deplete the eukaryotic host DNA. Protocols for effective depletion of coral host DNA and enrichment of bacterial DNA coverage for application in metagenome shotgun sequencing studies to our knowledge do not exist (Neave, Michell, Apprill, & Voolstra, 2017; Robbins et al., 2019). To address this, we here developed a protocol for coral DNA preservation and DNA extraction suitable for host- (e.g., sWGS), marker gene- (e.g., ITS2, 16S), and metagenomicsbased (e.g., microbiome) sequencing approaches. The protocol employs the DNAand cell morphology-/integrity-preserving properties of DESS buffer, compatible with application in remote field settings and evading the need for cooling/freezing of samples. Sprayed-off tissue from coral fragments is then used for DNA extraction using two distinct extraction kits (the Qiagen DNeasy Blood & Tissue Kit and the Qiagen QIAamp DNA Microbiome Kit) for use in host-, marker gene-, and metagenomics-based sequencing approaches. The protocol was successfully applied to coral samples from Acropora sp., Pocillopora sp., and Porites sp.. A detailed study on performance, efficacy, and benchmarking of the here-developed protocol in comparison to a range of other protocols is in preparation and is anticipated to be available shortly. We hope the here-developed protocol applies broadly and will be used widely. 2 Protocol (step-by-step): DESS buffer-based sample collection, storage, and processing for DNA isolation of coral field samples Resources/Read before you start - DNeasy Blood & Tissue Handbook - QIAamp DNA Microbiome Handbook Coral sample collection 1. Collect branching coral species (e.g., Acropora sp.) with hammer and chisel or clippers, collect massive coral species (e.g., Porites sp.) with underwater drill and core drill bit (e.g., link) or hammer and chisel. NOTE: In our experience small fragments (~2 cm2 surface) yield sufficient DNA for host-, marker gene-, and metagenomics-based sequencing approaches (DNeasy kit: ~100 ng/µL in 50 µL; QIAamp kit: ~10 ng/µL in 50 µL). 2. Place specimens into collection bags, make sure the bags are filled with seawater. 3. Upon return to the boat/shore, place the sample bags inside a cooler box filled with seawater until further processing. DESS buffer-based coral sample storage Long-term storage: 1. Transfer coral sample from sample bag into 5 mL (e.g., link) or 25 mL (e.g., link) tubes (wider tube opening makes it easier to fit coral fragments!) and fill up with DESS buffer, so the fragment is fully immersed/covered. 2. Incubate for ~2 hours at RT, store indefinitely at RT or 4 °C (if possible keep cool, but not critical if specimens are not cooled at all times). NOTE: Do NOT freeze, as this will break cells, impeding ability to differentially lyse eukaryotic/prokaryotic cells (important for metagenomics downstream processing). NOTE: Seal all tubes with parafilm before shipping/transport. Short-term storage: 1. Transfer coral sample from sample bag into whirl-pak bags (e.g., link) and add 5 mL of DESS buffer to fully immerse the fragment. NOTE: in our experience, 2 mL of DESS buffer are sufficient to cover a small fragment, hence increasing DNA concentration. 2. Use a reusable cable tie to seal the whirl-pak bag. 3. Incubate for ~2 hours at RT, store at RT or 4 °C until further processing. NOTE: Do NOT freeze, as this will break cells, inferring ability to differentially lyse eukaryotic/prokaryotic cells, important for metagenomics downstream processing). 3 DESS buffer recipe - Final solution: 0.25 M EDTA, 20 % DMSO, 150 - 200 g NaCl / 1000 mL. - Recipe for 1 L DESS: - 500 mL 0.5 M EDTA pH 8 (e.g., from AppliChem) - 200 mL DMSO - 300 mL H2O - 150 g NaCl - Not all NaCl will be dissolved; a hypersaturated solution has precipitated NaCl at the bottom. Coral sample processing & storage 1. Decant DESS buffer for volumes ≥ 5 mL For DESS buffer volumes ≤ 2 mL, directly proceed to step 2. 2. Spray off coral tissue into the short-term storage whirl-pak bag using airflow from a sterile 1000 µL pipette filter-tip connected via a rubber hose to a bench top air pressure valve or an airbrush compressor set, until all coral tissue is removed from the skeleton. NOTE: For DESS buffer volumes ≥5 mL, we use the ‘wet’ coral sample; the DESS buffer remaining on the coral sample is sufficient to spray off tissue. For DESS buffer volumes ≤2 mL, it is more efficient to directly spray off tissue into the buffer and transfer into storage tube (step 3). 3. Transfer tissue slurry into a screw-cap cryogenic vial (e.g., link; cat no E316.1) and vortex (typical volume for ‘wet’ coral sample is 100 - 1000 µL, depending on size of fragment; typical volume is 2 mL if initial DESS buffer volume is kept). NOTE: Store at RT or 4°C (i.e., for differential lysis to work for microbiome DNA isolation, tissue cannot be frozen! See below) 4 DNA isolation using the DNeasy Blood & Tissue Kit with RNA digestion for use in hostand marker gene-based sequencing approaches (e.g., sWGS, 16S, ITS2) 1. Transfer the tissue slurry into a 2 ml microtube, centrifuge at 10,000 x g for 3 min at RT to pellet tissue slurry 2. Pipet off supernatant DESS buffer so that ~700-800µl remain in the tube. 3. Homogenize the tissue slurry in remaining DESS with a homogenizer (e.g., Polytron PT 1200 E with a 3 mm dispersing aggregate, e.g. link). NOTE: Incomplete homogenization leads to significantly reduced DNA yields and can cause clogging of the spin column. NOTE: Store the remaining homogenized sample in DESS at RT or +4°C 4. Transfer 90 µL of homogenized tissue slurry from above (‘Preparations for DNA isolation’) into a 1.5 mL tube. NOTE: We follow the procedures based on the “Protocol: Purification of Total DNA from Animal Tissues” and the “Optional: RNase A-based RNA digestion procedure.” NOTE: All steps are at RT unless otherwise indicated. 5. Add 90 µL of Buffer ATL and 20 µL proteinase K (= 200 µL total volume). 6. Mix by vortexing and incubate at 56 °C for 1 h (until completely lysed); mix occasionally during incubation (e.g., by using a thermomixer). 4. Add 4 µL RNase A (100 mg/mL, link), mix by vortexing, and incubate for 2 min. 5. Vortex for 15 sec. Add 200 µL Buffer AL. Mix thoroughly by vortexing. 6. Add 200 µL ethanol (96–100%). Mix thoroughly by vortexing. 7. Pipet the mixture into a DNeasy Mini spin column placed in a 2 mL collection tube. Centrifuge at ≥ 6000 x g for 1 min. Discard the flow-through and collection tube. 8. Place the spin column in a new 2 mL collection tube. Add 500 µL Buffer AW1. Centrifuge for 1 min at ≥ 6000 x g. Discard the flow-through and collection tube. 9. Place the spin column in a new 2 mL collection tube, add 500 µL Buffer AW2 and centrifuge for 3 min at 20,000 x g. Discard the flow-through. Reuse the collection tube. 10. Place the spin column in the 2 mL collection tube. Centrifuge at full speed for 1 min to dry the membrane. 11. Transfer the spin column to a new 1.5 mL tube. 12. Elute the DNA by adding 30 µL Buffer AE to the center of the spin column membrane. Incubate for 1 min. Centrifuge for 1 min at ≥ 6000 x g. 13. Assess DNA quantity and quality; store at -20°C. 5 DNA isolation using the QIAGEN Microbiome Kit for use in metagenomics-based sequencing approaches 1. Transfer the tissue slurry into a 2 ml microtube and centrifuge at 10,000 x g for 3 min at RT to pellet tissue slurry. 2. Pipet off supernatant DESS buffer completely, then wash the pellet with 1 mL 1 x PBS buffer. Vortex briefly. 3. Centrifuge at 10,000 x g for 3 min at RT to pellet tissue slurry and discard the supernatant. 4. Add 1 mL 1 x PBS buffer and homogenize the tissue slurry with a homogenizer (e.g., Polytron PT 1200 E with a 3 mm dispersing aggregate, e.g. link). NOTE: Incomplete homogenization leads to significantly reduced DNA yields and can cause clogging of the spin column. NOTE: The homogenized tissue cannot be stored without losing compatibility with the protocol. Only homogenize the tissue intended for immediate extraction. 5. Centrifuge the sample at 300 x g for 2 minutes. 6. Carefully transfer the supernatant to a new 2 mL tube and discard the pellet. If necessary, re-adjust the volume to 1 mL with 1 x PBS buffer, following transfer. NOTE: This centrifugation step aims to pellet Symbiodiniaceae cells. Reducing Symbiodiniaceae cells in the tissue slurry is important for the efficient recovery of prokaryotic DNA. The centrifugation speed and time were chosen to minimize the pelleting of prokaryotic cells. NOTE: The pellet will be soft. Be extra careful. NOTE: We follow the procedures based on the “Protocol: Depletion of host DNA" from the QIAamp DNA Microbiome Handbook. NOTE: All steps are at RT unless otherwise indicated. 7. Add 500 µL Buffer AHL to the 1000 µL homogenized tissue and transfer to a new 2 mL tube. Optional bead-beating step: a. Move the homogenized tissue and the Buffer AHL to a QIAGEN PowerBead Tube, Ceramic 1.4 mm (link) b. Place the PowerBead Tube into a PowerLyzer24 Homogenizer (QIAGEN). Homogenize at 2,000 rpm for 2 x 30 sec with incubation on ice for 30 sec. inbetween. c. Transfer the homogenate to a new 2 mL tube. NOTE: Optional bead-beating step facilitates cracking/opening of Symbiodiniaceae cells, which improves lysis, i.e. efficient removal, of algal DNA. The bead material, size, speed, and time for bead-beating time were chosen to minimize the opening of prokaryotic cells. 6 This step is affected by the quality of the initial tissue slurry and may lead to increased damage to DNA fragments, resulting in shorter lengths. 8. Incubate for 30 min at RT with end-over-end rotation (e.g., on a rotating wheel). 9. Centrifuge at 10,000 x g for 10 min. 10. Carefully remove the supernatant without disturbing the pellet. 11. Add 190 µl Buffer RDD and 2.5 µL Benzonase. Mix well and incubate at 37 °C for 30 min at 600 rpm in a thermomixer. 12. Add 20 μL Proteinase K, vortex, and incubate at 56 °C for 30 min at 600 rpm in a thermomixer. 13. Spin the tube at slow speed to remove condensation. 14. Add 200 µL Buffer ATL (containing reagent DX). Mix well and transfer into a Pathogen Lysis Tube L. 15. Place the Pathogen Lysis Tube L into a PowerLyzer24 Homogenizer (Qiagen). Homogenize at 4,000 rpm for 45 sec and incubate on ice for 5 min. Repeat for a total of two times. NOTE: This bead-beating step targets prokaryotic cells; the Pathogen Lysis Tube L contains the beads already. Other options exist, please refer to the handbook/manual. 16. Centrifuge Pathogen Lysis Tube L at 10,000 x g for 1 min to reduce foam after lysis. 17. Mix carefully by pipetting up and down (without re-suspending the beads) and transfer the supernatant to a fresh 1.5 mL tube. NOTE: Do not transfer beads from the Pathogen Lysis Tube L to subsequent reactions. 18. Add 40 µL Proteinase K, vortex, and incubate at 56 °C for 30 min at 600 rpm in a thermomixer. 19. Add 200 µL Buffer APL2, vortex for 15 - 30 sec, and incubate at 70 °C for 10 min in a thermomixer. 20. Briefly spin the tube and add 200 µL ethanol. Vortex for 15 - 30 sec. 21. Transfer up to 700 µL of the mixture into a QIAmp UCP Mini Column placed in a 2 mL collection tube. 22. Centrifuge at ≥ 6000 x g for 1 min. Discard the flow-through and repeat the previous step with any remaining mixture. 23. Place the spin column in a new 2 mL collection tube, add 500 µL Buffer AW1. Centrifuge at ≥ 6000 x g for 1 min. Discard the flow-through and collection tube. 24. Place the spin column in a new 2 mL collection tube, add 500 µL Buffer AW2, and centrifuge for 3 min at 20,000 x g. Discard the flow-through and collection tube. 25. Place the spin column in a new 2 mL collection tube. Centrifuge at full speed for 1 min to dry the membrane. 26. Transfer the spin column to a new 1.5 mL elution tube. 27. Add 50 µL Buffer AVE directly to the center of the spin column membrane. 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